Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador.

INTRODUCTION: Carbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022. METHODS: Ninety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC, blaNDM, blaOXA-48, blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes. RESULTS: Carbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases. CONCLUSION: The epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

An Emergent Change in Epidemiologic and Microbiological Characteristics of Bloodstream Infections in Adults With Febrile Neutropenia Resulting From Chemotherapy for Acute Leukemia and Lymphoma at Reference Centers in Chile, Ecuador, and Peru.

Background: Febrile neutropenia is a life-threatening condition commonly observed in patients with hematologic malignancies. The aim of this article is to provide updated knowledge about bloodstream infections in febrile neutropenia episodes within the Andean region of Latin America. Method: This retrospective study was based in 6 hospitals in Chile, Ecuador, and Peru and included adult patients with acute leukemia or lymphoma and febrile neutropenia between January 2019 and December 2020. Results: Of the 416 febrile neutropenia episodes, 38.7% had a bloodstream infection, 86% of which were caused by gram-negative rods, with Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa being the most frequently identified bacteria. K pneumoniae isolates were more frequently resistant than E coli to cefotaxime (65% vs 39.6%), piperacillin-tazobactam (56.7% vs 27.1%), and imipenem (35% vs 2.1%) and were more frequently multidrug resistant (61.7% vs 12.5%). Among P aeruginosa, 26.7% were resistant to ceftazidime, piperacillin-tazobactam, and imipenem, and 23.3% were multidrug resistant. Overall 30-day mortality was 19.8%, being higher with vs without a bloodstream infection (26.7% vs 15.3%, P = .005). Fever duration was also significantly longer, as well as periods of neutropenia and length of hospital stay for patients with bloodstream infection. Additionally, the 30-day mortality rate was higher for episodes with inappropriate vs appropriate empirical antibiotic therapy (41.2% vs 26.6%, P = .139). Conclusions: Considering the high rates of bacteria-resistant infection and 30-day mortality, it is imperative to establish strategies that reduce the frequency of bloodstream infections, increasing early identification of patients at higher risks of multidrug bacteria resistance, and updating existing empirical antibiotic recommendations.

Colistin resistance screening by 3 µg/ml colistin agar in Carbapenemase-producing Enterobacterales.

BACKGROUND: In low- and middle-income countries, the use of colistin in therapeutic regimens is common, to treat infections produced for Carbapenemase-producing Enterobacterales (CPE) due to limited access to the recently discovered-approved antibiotics. Furthermore, the technical limitations to perform colistin susceptibility tests make it difficult to assess the suitability of this treatment for each patient, as well as to monitor the rates of resistance. In the present study, we describe the use of agar dilution using a unique colistin concentration of 3 µg/ml to discriminate isolates with colistin resistance in CPE obtained from clinical samples. METHODS: Clinical Laboratory Standards Institute (CLSI) colistin broth microdilution method and dilution agar with a colistin concentration of 3 µg/ml were performed in 168 isolates of CPE obtained from clinical samples in Guayaquil, Ecuador. Broth microdilution was considered our gold standard using CLSI breakpoints as reference (≤2 µg/ml intermediate and ≥4 µg/ml resistant). Categorical agreement was defined as obtaining a reading within the same category with both methodologies. RESULTS: Isolates obtained from respiratory samples were the most prevalent (26.19%; n = 44). Klebsiella pneumoniae was the predominant specie (94.04%; n = 158). KPC-like carbapenemase was present in all the isolates, and interestingly, colistin resistance was not mediated by MCR-1 production. Categorical agreement between both methods resulted in 97.02%. CONCLUSION: We propose the use of dilution agar with a colistin concentration of 3 µg/ml, as a valid method for screening colistin resistance in low- and middle-income countries to monitor resistance and to perform epidemiological studies.

Macrolides: a novel risk factor for carbapenemase-producing Enterobacterales in intensive care units.

INTRODUCTION: Carbapenemase-producing Enterobacterales (CPE) have emerged as a substantial cause of morbi-mortality worldwide, with a prevalence of approximately 5% in areas with high endemicity. However, available data may not be representative of developing countries, such as Ecuador. In this study, the incidence of CPE in Ecuador and risk factors for infection/colonisation were evaluated. METHODOLOGY: A prospective cohort study was performed from February to April 2016 in seven intensive-care units of Guayaquil, Ecuador. Samples were processed according to the Centers for Disease Control and Prevention laboratory protocol and the CHROMagar mSuper CARBA agar method. Resistance to carbapenems was defined according to Clinical and Laboratory Standards Institute breakpoints. A modified carbapenemase inactivation method was used to identify carbapenamase production phenotypically with molecular confirmation by multiplex polymerase chain reaction. RESULTS: In total, 640 patients were enrolled. The incidence of CPE was 36.4% (N = 233). A multivariate analysis indicated that several factors were associated with CPE acquisition, included a long intensive care unit stay (OR 1.05; 95% CI 1.03-1.08; p < 0.01), tracheostomy (OR 3.52; 95% CI 1.90-6.75; p < 0.01), hospitalisation 3 months prior to admission (OR 2.07; 95% CI 1.17-3.71; p < 0.01), vancomycin use (OR 3.31; 95% CI 2.02-5.18; p < 0.01), and macrolide use (OR 3.31; 95% CI 1.43-7.76; p < 0.01). CONCLUSIONS: Macrolide use was a risk factor for CPE acquisition. This association should be evaluated further, especially in developing countries.

Zika virus infection in pregnancy: a protocol for the joint analysis of the prospective cohort studies of the ZIKAlliance, ZikaPLAN and ZIKAction consortia.

INTRODUCTION: Zika virus (ZIKV) infection in pregnancy has been associated with microcephaly and severe neurological damage to the fetus. Our aim is to document the risks of adverse pregnancy and birth outcomes and the prevalence of laboratory markers of congenital infection in deliveries to women experiencing ZIKV infection during pregnancy, using data from European Commission-funded prospective cohort studies in 20 centres in 11 countries across Latin America and the Caribbean. METHODS AND ANALYSIS: We will carry out a centre-by-centre analysis of the risks of adverse pregnancy and birth outcomes, comparing women with confirmed and suspected ZIKV infection in pregnancy to those with no evidence of infection in pregnancy. We will document the proportion of deliveries in which laboratory markers of congenital infection were present. Finally, we will investigate the associations of trimester of maternal infection in pregnancy, presence or absence of maternal symptoms of acute ZIKV infection and previous flavivirus infections with adverse outcomes and with markers of congenital infection. Centre-specific estimates will be pooled using a two-stage approach. ETHICS AND DISSEMINATION: Ethical approval was obtained at each centre. Findings will be presented at international conferences and published in peer-reviewed open access journals and discussed with local public health officials and representatives of the national Ministries of Health, Pan American Health Organization and WHO involved with ZIKV prevention and control activities.

Comparación de dos métodos comerciales para la determinación de la concentración mínima inhibitoria de meropenem en Klebsiella pneumoniae productora de carbapenemasa tipo KPC / Comparison of two commercial methods for determination of the minimum inhibitory meropenem concentration in KPC carbapenemase-producing Klebsiella pneumoniae

Introducción: El tratamiento de las infecciones por Klebsiella pneumoniae productora de carbapenemasa tipo KPC es complicado debido a las escasas opciones terapéuticas existentes, lo cual obliga a optimizar los esquemas terapéuticos disponibles. Objetivo: Determinar la concordancia de la tarjeta AST-N272 del Sistema Vitek 2 Compact y las tiras M.I.C.ETM Evaluator con la dilución en agar para la determinación de la concentración mínima inhibitoria del meropenem en Klebsiella pneumoniae productora de carbapenemasa tipo KPC. Métodos: Se estudiaron 53 aislados de K. pneumoniae bla KPC positivas no clonales, provenientes de hisopados rectales recolectados en diferentes unidades hospitalarias de Guayaquil, Ecuador, entre enero a junio de 2016. Se determinó la concentración mínima inhibitoria de meropenem por dilución en agar (método de referencia), así como por el sistema Vitek 2 Compact (AST-N272) y las tiras M.I.C.ETM. Se determinó la CMI 50, CMI 90 y la concordancia esencial. Resultados: El rango de la CMI de meropenem de los aislados estudiados fue de 1 a ≥ 32 µg/mL, con una CMI50= 4 µg/mL y una CMI90= ≥ 32 µg/mL. El 86,79 por ciento (n= 46) de los aislados tuvo una CMI≤ 8 µg/mL. Se observó un 94,33 por ciento de concordancia esencial con las tiras M.I.C.ETM, mientras que la tarjeta AST-N272 mostró una concordancia esencial inferior al 50 por ciento. Conclusiones: Los resultados sugieren posibles implicaciones en el tratataminto del paciente, pues reduce opciones terapéuticas en contextos de difícil manejo. Además, resaltan la necesidad de la confirmación de la resistencia a carbapenémicos mediante el método de Kirby Bawer en aquellos laboratorios que tienen métodos automatizados para estudios de susceptibilidad(AU)

Introduction: The treatment for KPC carbapenemase-producing Klebsiella pneumoniae infections is complicated, due to the scant therapeutic options available, which forces us to optimize the therapies at hand. Objective: Determine the agreement between the AST-N272 card of the Vitek 2 Compact system and the M.I.C.E.TM Evaluator strips, and the agar dilution method for determination of the minimum inhibitory meropenem concentration in KPC carbapenemase-producing Klebsiella pneumoniae. Methods: A study was conducted of 53 positive non-clonal K. pneumoniae bla KPC isolates from rectal swabs collected at several hospitals in Guayaquil, Ecuador, from January to June 2016. Minimum inhibitory meropenem concentration was determined by agar dilution (reference method), the Vitek 2 Compact system (AST-N272) and M.I.C.E.TM strips. Determination was made of MIC 50, MIC 90 and essential agreement. Results: The meropenem MIC range for the isolates studied was 1 to ≥ 32 µg/ml, with MIC50= 4 µg/ml and MIC90= ≥ 32 µg/ml. In 86.79 percent (n= 46) of the isolates MIC was ≤ 8 µg/ml. Essential agreement was 94.33 percent with the M.I.C.E.TM strips and under 50 percent with the AST-N272 card. Conclusions: The results obtained suggest potential implications for the treatment of patients, since therapeutic options are reduced in difficult management contexts. They also highlight the need for confirmation of carbapenem resistance by the Kirby-Bauer procedure in laboratories equipped with automated methods for susceptibility studies(AU)

Carbapenemase producing Enterobacteriaceae in intensive care units in Ecuador: Results from a multicenter study.

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae (CPE) are of global concern due to the growing number of patients who acquire them and their association with high mortality rates. Although there are some reports of endemicity in developing countries, little is known about this microorganism, and Ecuador is not an exception. Subsequently, our objective was to clinically and molecularly characterize carbapenemase producing-Enterobacteriaceae in intensive care units (ICUs) in Guayaquil, Ecuador. METHODS: To determine CPE colonization, we obtained perineal and inguinal swabs from patients admitted to seven intensive-care adult units in Guayaquil-Ecuador between February and April 2016. The Centers for Disease Control and Prevention (CDC) laboratory protocol and chromogenic agar were used to process the cultures. Polymerase chain reaction was used to confirm carbapenemase production. Genotypic analysis was performed by Multilocus Sequence Typing (MLST) and pulsed-field electrophoresis (PFEG). Demographic and clinical data were obtained from the electronic charts and patient's relatives. RESULTS: Six hundred seventy-seven patients were included in the study, of whom 255 were colonized/infected by CPE. The CPE prevalence was 37.67%. Previous use of antimicrobials, use of invasive procedures and being burned at admission were associated with CPE. The most frequent infection was found after a surgical procedure. Klebsiella pneumoniae (n=249) was the predominant microorganism harbouring blaKPC, followed by Enterobactercloacae (n=8), Klebsiella aerogenes (n=4), Escherichia coli (n=4) and Klebsiella oxytoca (n=1). NDM was present in Proteus mirabilis. The strains were distributed in 19 sequence types (ST), and 10 were not reported previously in Ecuador. ST 258 was the sequence type isolated most frequently. CONCLUSION: This study shows a high prevalence of CPE in ICUs, particularly K. pneumoniae blaKPC ST 258. The identification of KPC alleles may help to understand the routes of dissemination and control spread within ICUs in Guayaquil, Ecuador.

Tracking KPC-3-producing ST-258 Klebsiella pneumoniae outbreak in a third-level hospital in Granada (Andalusia, Spain) by risk factors and molecular characteristics.

The objective of this study was to determine clinical-epidemiological characteristics of the patients and the genetic characteristics of carbapenemase KPC-3-producing Klebsiella pneumoniae isolates belonging to sequence type ST258. The eligible study population was all patients with isolates detected between October 2015 and March 2017. Clinical-epidemiological and microbiological data were gathered on risk factors associated with infection by this clone. Antimicrobial susceptibility was determined using MicroScan system and diffusion in agar. Genes encoding carbapenemases were detected using PCR and Sanger sequencing. The sequence type was assigned by MLST, and the genetic relationship among clinical isolates was determined by pulsed field electrophoresis and by analysis of the genetic environment. The study included 23 individuals with isolates of KPC-3/ST258; the mean age was 77 year, and mean stay pre-isolation was 32 days; 81% received empirical antimicrobial treatment. Isolates were only susceptible to gentamicin (CIM ≤ 2 mg/L), tigecycline (CIM ≤ 1 mg/L), and colistin (CIM ≤ 2 mg/L). The isolates belonged to ST258, with five pulse types or subgroups. All isolates showed amplification of KPC, which was identified as KPC-3 variant. Gene blaKPC-3 was flanked by insertion sequences Kpn6 and Kpn7 within Tn4401 transposon isoform a. We report, for the first time in Spain, an 18-month outbreak by KPC-3-producing ST258 K. pneumoniae. Its acquisition was associated with a history of antimicrobial therapy, with three treatment options, and with high mortality. The detection of different pulse types is attributable to different introductions of the clone in our setting, supporting the need for multi-resistant isolate surveillance studies.

Understanding the relation between Zika virus infection during pregnancy and adverse fetal, infant and child outcomes: a protocol for a systematic review and individual participant data meta-analysis of longitudinal studies of pregnant women and their infants and children.

INTRODUCTION: Zika virus (ZIKV) infection during pregnancy is a known cause of microcephaly and other congenital and developmental anomalies. In the absence of a ZIKV vaccine or prophylactics, principal investigators (PIs) and international leaders in ZIKV research have formed the ZIKV Individual Participant Data (IPD) Consortium to identify, collect and synthesise IPD from longitudinal studies of pregnant women that measure ZIKV infection during pregnancy and fetal, infant or child outcomes. METHODS AND ANALYSIS: We will identify eligible studies through the ZIKV IPD Consortium membership and a systematic review and invite study PIs to participate in the IPD meta-analysis (IPD-MA). We will use the combined dataset to estimate the relative and absolute risk of congenital Zika syndrome (CZS), including microcephaly and late symptomatic congenital infections; identify and explore sources of heterogeneity in those estimates and develop and validate a risk prediction model to identify the pregnancies at the highest risk of CZS or adverse developmental outcomes. The variable accuracy of diagnostic assays and differences in exposure and outcome definitions means that included studies will have a higher level of systematic variability, a component of measurement error, than an IPD-MA of studies of an established pathogen. We will use expert testimony, existing internal and external diagnostic accuracy validation studies and laboratory external quality assessments to inform the distribution of measurement error in our models. We will apply both Bayesian and frequentist methods to directly account for these and other sources of uncertainty. ETHICS AND DISSEMINATION: The IPD-MA was deemed exempt from ethical review. We will convene a group of patient advocates to evaluate the ethical implications and utility of the risk stratification tool. Findings from these analyses will be shared via national and international conferences and through publication in open access, peer-reviewed journals. TRIAL REGISTRATION NUMBER: PROSPERO International prospective register of systematic reviews (CRD42017068915).

Aplicación y aceptabilidad de la Guía Clínica de Dengue OMS-2009: la percepción de Ecuador / Aplicability and Aceptability and of the Dengue Clinical Guidelines WHO 2009: Ecuador's perception

Introducción: la infección por virus dengue se ha diseminado a más de 100 países a nivel mundial, con un cambio en su patrón que hizo que la clasificación OMS 1997 no sea aplicable. Objetivo: determinar la aceptabilidad y aplicabilidad de la guía OMS-2009. Materiales y métodos: fue conducido un estudio de corte transversal con diseño mixto, entre abril a noviembre del 2009 posterior a una intervención formativa dirigida a médicos, para lo cual se aplicó un cuestionario previamente elaborado y se realizó un trabajo de grupos focales. Resultados: los médicos mostraron una aceptación del 91,9% por la clasificación OMS-2009. El 100% realiza monitoreo de los signos vitales, considerando plaquetas (98,4%) y hematocrito (96.9%). El 96,9% utiliza las plaquetas como signos de alarma, seguido de letargia o inquietud (95,3%), dolor abdominal (95,3%), vómitos persistentes (92,2%). Los participantes refieren que es útil en la clasificación de los pacientes, especialmente los graves (30,56%), fácil de entender y manejar (30,56%), y como una ventaja que ayuda en el manejo y tratamiento de los casos (44,4%). El 80% considera que es necesaria su difusión a través de capacitaciones. De los grupos focales se considera que es sencilla, practica y didáctica para los niveles de severidad, orienta al triage de los pacientes y permite mejor su clasificación. Conclusiones: la guía OMS-2009 tiene una amplia aceptación por el personal de salud (AU).

Introduction: dengue virus infection has spread to more than 100 countries worldwide, with a change in its pattern that made the 1997 WHO classification not applicable. Objective: to determine the acceptability and applicability of the WHO-2009 guide. Materials and methods: a cross-sectional study with a mixed design was conducted between April and November 2009 following a training intervention directed at physicians, for which a previously elaborated questionnaire was applied and a focus group work was carried out. Results: physicians showed an acceptance of 91.9% for the WHO-2009 classification. 100% performed vital signs monitoring, considering platelets (98.4%) and hematocrit (96.9%). 96.9% used platelets as warning signs, followed by lethargy or restlessness (95.3%), abdominal pain (95.3%), persistent vomiting (92.2%). Participants report that it is useful in the classification of patients, especially severe (30.56%), easy to understand and manage (30.56%), and as an aid to the management and treatment of cases (44.4%). Eighty percent consider it necessary to disseminate it through training. Conclusions: gráficothe WHO-2009 guide has a wide acceptance by the health personnel, the health professionals, the health professionals, the health professionals (AU).

Aplicación y aceptabilidad de la Guía Clínica de Dengue OMS-2009: la percepción de Ecuador / Aplicability and Aceptability and of the Dengue Clinical Guidelines WHO 2009: Ecuador's perception

Introducción: la infección por virus dengue se ha diseminado a más de 100 países a nivel mundial, con un cambio en su patrón que hizo que la clasificación OMS 1997 no sea aplicable. Objetivo: determinar la aceptabilidad y aplicabilidad de la guía OMS-2009. Materiales y métodos: fue conducido un estudio de corte transversal con diseño mixto, entre abril a noviembre del 2009 posterior a una intervención formativa dirigida a médicos, para lo cual se aplicó un cuestionario previamente elaborado y se realizó un trabajo de grupos focales. Resultados: los médicos mostraron una aceptación del 91,9% por la clasificación OMS-2009. El 100% realiza monitoreo de los signos vitales, considerando plaquetas (98,4%) y hematocrito (96.9%). El 96,9% utiliza las plaquetas como signos de alarma, seguido de letargia o inquietud (95,3%), dolor abdominal (95,3%), vómitos persistentes (92,2%). Los participantes refieren que es útil en la clasificación de los pacientes, especialmente los graves (30,56%), fácil de entender y manejar (30,56%), y como una ventaja que ayuda en el manejo y tratamiento de los casos (44,4%). El 80% considera que es necesaria su difusión a través de capacitaciones. De los grupos focales se considera que es sencilla, practica y didáctica para los niveles de severidad, orienta al triage de los pacientes y permite mejor su clasificación. Conclusiones: la guía OMS-2009 tiene una amplia aceptación por el personal de salud (AU).

Introduction: dengue virus infection has spread to more than 100 countries worldwide, with a change in its pattern that made the 1997 WHO classification not applicable. Objective: to determine the acceptability and applicability of the WHO-2009 guide. Materials and methods: a cross-sectional study with a mixed design was conducted between April and November 2009 following a training intervention directed at physicians, for which a previously elaborated questionnaire was applied and a focus group work was carried out. Results: physicians showed an acceptance of 91.9% for the WHO-2009 classification. 100% performed vital signs monitoring, considering platelets (98.4%) and hematocrit (96.9%). 96.9% used platelets as warning signs, followed by lethargy or restlessness (95.3%), abdominal pain (95.3%), persistent vomiting (92.2%). Participants report that it is useful in the classification of patients, especially severe (30.56%), easy to understand and manage (30.56%), and as an aid to the management and treatment of cases (44.4%). Eighty percent consider it necessary to disseminate it through training. Conclusions: gráficothe WHO-2009 guide has a wide acceptance by the health personnel, the health professionals, the health professionals, the health professionals (AU).

The High-Quality Genome Sequence of the Oceanic Island Endemic Species Drosophila guanche Reveals Signals of Adaptive Evolution in Genes Related to Flight and Genome Stability.

Drosophila guanche is a member of the obscura group that originated in the Canary Islands archipelago upon its colonization by D. subobscura. It evolved into a new species in the laurisilva, a laurel forest present in wet regions that in the islands have only minor long-term weather fluctuations. Oceanic island endemic species such as D. guanche can become model species to investigate not only the relative role of drift and adaptation in speciation processes but also how population size affects nucleotide variation. Moreover, the previous identification of two satellite DNAs in D. guanche makes this species attractive for studying how centromeric DNA evolves. As a prerequisite for its establishment as a model species suitable to address all these questions, we generated a high-quality D. guanche genome sequence composed of 42 cytologically mapped scaffolds, which are assembled into six super-scaffolds (one per chromosome). The comparative analysis of the D. guanche proteome with that of twelve other Drosophila species identified 151 genes that were subject to adaptive evolution in the D. guanche lineage, with a subset of them being involved in flight and genome stability. For example, the Centromere Identifier (CID) protein, directly interacting with centromeric satellite DNA, shows signals of adaptation in this species. Both genomic analyses and FISH of the two satellites would support an ongoing replacement of centromeric satellite DNA in D. guanche.

Molecular population genetics of the Polycomb genes in Drosophila subobscura.

Polycomb group (PcG) proteins are important regulatory factors that modulate the chromatin state. They form protein complexes that repress gene expression by the introduction of posttranslational histone modifications. The study of PcG proteins divergence in Drosophila revealed signals of coevolution among them and an acceleration of the nonsynonymous evolutionary rate in the lineage ancestral to the obscura group species, mainly in subunits of the Pcl-PRC2 complex. Herein, we have studied the nucleotide polymorphism of PcG genes in a natural population of D. subobscura to detect whether natural selection has also modulated the evolution of these important regulatory genes in a more recent time scale. Results show that most genes are under the action of purifying selection and present a level and pattern of polymorphism consistent with predictions of the neutral model, the exceptions being Su(z)12 and Pho. MK tests indicate an accumulation of adaptive changes in the SU(Z)12 protein during the divergence of D. subobscura and D. guanche. In contrast, the HKA test shows a deficit of polymorphism at Pho. The most likely explanation for this reduced variation is the location of this gene in the dot-like chromosome and would indicate that this chromosome also has null or very low recombination in D. subobscura, as reported in D. melanogaster.

Sex combs reduced (Scr) regulatory region of Drosophila revisited.

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.

Evidence of neofunctionalization after the duplication of the highly conserved Polycomb group gene Caf1-55 in the obscura group of Drosophila.

Drosophila CAF1-55 protein is a subunit of the Polycomb repressive complex PRC2 and other protein complexes. It is a multifunctional and evolutionarily conserved protein that participates in nucleosome assembly and remodelling, as well as in the epigenetic regulation of a large set of target genes. Here, we describe and analyze the duplication of Caf1-55 in the obscura group of Drosophila. Paralogs exhibited a strong asymmetry in evolutionary rates, which suggests that they have evolved according to a neofunctionalization process. During this process, the ancestral copy has been kept under steady purifying selection to retain the ancestral function and the derived copy (Caf1-55dup) that originated via a DNA-mediated duplication event ~18 Mya, has been under clear episodic selection. Different maximum likelihood approaches confirmed the action of positive selection, in contrast to relaxed selection, on Caf1-55dup after the duplication. This adaptive process has also taken place more recently during the divergence of D. subobscura and D. guanche. The possible association of this duplication with a previously detected acceleration in the evolutionary rate of three CAF1-55 partners in PRC2 complexes is discussed. Finally, the timing and functional consequences of the Caf1-55 duplication is compared to other duplications of Polycomb genes.

Dense gene physical maps of the non-model species Drosophila subobscura.

The comparative analysis of genetic and physical maps as well as of whole genome sequences had revealed that in the Drosophila genus, most structural rearrangements occurred within chromosomal elements as a result of paracentric inversions. Genome sequence comparison would seem the best method to estimate rates of chromosomal evolution, but the high-quality reference genomes required for this endeavor are still scanty. Here, we have obtained dense physical maps for Muller elements A, C, and E of Drosophila subobscura, a species with an extensively studied rich and adaptive chromosomal polymorphism. These maps are based on 462 markers: 115, 236, and 111 markers for elements A, C, and E, respectively. The availability of these dense maps will facilitate genome assembly and will thus greatly contribute to obtaining a good reference genome, which is a required step for D. subobscura to attain the model species status. The comparative analysis of these physical maps and those obtained from the D. pseudoobscura and D. melanogaster genomes allowed us to infer the number of fixed inversions and chromosomal evolutionary rates for each pairwise comparison. For all three elements, rates inferred from the more closely related species were higher than those inferred from the more distantly related species, which together with results of relative-rate tests point to an acceleration in the D. subobscura lineage at least for elements A and E.

Inferring the demographic history of Drosophila subobscura from nucleotide variation at regions not affected by chromosomal inversions.

Drosophila subobscura presents a rich and complex chromosomal inversion polymorphism. It can thus be considered a model system (i) to study the mechanisms originating inversions and how inversions affect the levels and patterns of variation in the inverted regions and (ii) to study adaptation at both the single-gene and chromosomal inversion levels. It is therefore important to infer its demographic history as previous information indicated that its nucleotide variation is not at mutation-drift equilibrium. For that purpose, we sequenced 16 noncoding regions distributed across those parts of the J chromosome not affected by inversions in the studied population and possibly either by other selective events. The pattern of variation detected in these 16 regions is similar to that previously reported within different chromosomal arrangements, suggesting that the latter results would, thus, mainly reflect recent demographic events rather than the partial selective sweep imposed by the origin and frequency increase of inversions. Among the simple demographic models considered in our Approximate Bayesian Computation analysis of variation at the 16 regions, the model best supported by the data implies a population size expansion soon after the penultimate glacial period. This model constitutes a better null model, and it is therefore an important resource for subsequent studies aiming among others to uncover selective events across the species genome. Our results also highlight the importance of introducing the possibility of multiple hits in the coalescent simulations with an outgroup.

Characterization of the breakpoints of a polymorphic inversion complex detects strict and broad breakpoint reuse at the molecular level.

Inversions are an integral part of structural variation within species, and they play a leading role in genome reorganization across species. Work at both the cytological and genome sequence levels has revealed heterogeneity in the distribution of inversion breakpoints, with some regions being recurrently used. Breakpoint reuse at the molecular level has mostly been assessed for fixed inversions through genome sequence comparison, and therefore rather broadly. Here, we have identified and sequenced the breakpoints of two polymorphic inversions-E1 and E2 that share a breakpoint-in the extant Est and E1 + 2 chromosomal arrangements of Drosophila subobscura. The breakpoints are two medium-sized repeated motifs that mediated the inversions by two different mechanisms: E1 via staggered breaks and subsequent repair and E2 via repeat-mediated ectopic recombination. The fine delimitation of the shared breakpoint revealed its strict reuse at the molecular level regardless of which was the intermediate arrangement. The occurrence of other rearrangements in the most proximal and distal extended breakpoint regions reveals the broad reuse of these regions. This differential degree of fragility might be related to their sharing the presence outside the inverted region of snoRNA-encoding genes.

Structure and population genetics of the breakpoints of a polymorphic inversion in Drosophila subobscura.

Drosophila subobscura is a paleartic species of the obscura group with a rich chromosomal polymorphism. To further our understanding on the origin of inversions and on how they regain variation, we have identified and sequenced the two breakpoints of a polymorphic inversion of D. subobscura--inversion 3 of the O chromosome--in a population sample. The breakpoints could be identified as two rather short fragments (∼300 bp and 60 bp long) with no similarity to any known transposable element family or repetitive sequence. The presence of the ∼300-bp fragment at the two breakpoints of inverted chromosomes implies its duplication, an indication of the inversion origin via staggered double-strand breaks. Present results and previous findings support that the mode of origin of inversions is neither related to the inversion age nor species-group specific. The breakpoint regions do not consistently exhibit the lower level of variation within and stronger genetic differentiation between arrangements than more internal regions that would be expected, even in moderately small inversions, if gene conversion were greatly restricted at inversion breakpoints. Comparison of the proximal breakpoint region in species of the obscura group shows that this breakpoint lies in a small high-turnover fragment within a long collinear region (∼300 kb).